Osteoblast Menin Regulates Bone Mass in Vivo*

Background: Constitutive homozygous Men1−/− mice at mid-gestation are small with craniofacial defects. Results: Conditional osteoblast Men1 knock-out mice have reduced bone mass, and transgenic osteoblast menin-overexpressing mice have increased bone mass. Conclusion: Knock-out mice menin-deficient primary osteoblasts have impaired mineralization and reduced responsiveness to TGF-β and BMP-2. Significance: Menin is a potential target for gain of function therapeutics in low bone mass disorders. Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.

Both TGF-␤ and BMPs, 6 primarily synthesized by osteoblasts, play an important role in bone formation and remodeling (1)(2)(3). Autocrine and paracrine stimulation by TGF-␤ itself is important for the maintenance and expansion of mesenchymal stem cells, the osteoblast progenitors, and is critical for osteoblast differentiation and bone formation. BMPs were initially identified by their ability to induce ectopic bone formation in muscle, and some of them are now approved for use in clinical applications related to long bone fracture repair. Members of the TGF-␤ ligand superfamily signal through type I and type II serine/threonine kinase cell membrane receptors. The type II receptor is constitutively phosphorylated and, when activated by binding of the ligand, recruits and transphosphorylates the type I receptor. The activated type I receptor phosphorylates the receptor-regulated Smads (R-Smad; Smad2 and Smad3 for TGF-␤ and Smad1, Smad5, and Smad8 for BMPs) inducing their association with the common partner, Smad4. The R-Smad⅐Smad4 complex then translocates to the nucleus to activate specific genes (4,5). The importance of TGF-␤ and BMPs in many components of the musculoskeletal system has been emphasized by the finding of mutations in their signaling pathway molecules in human skeletal genetic disorders.
The protein menin came to attention initially as the tumor suppressor encoded by the MEN1 gene that is implicated in the autosomal dominant multiple endocrine neoplasia type 1 (MEN1) disorder. In this syndrome tumors arise in select endocrine (and non-endocrine) tissues during the lifetime of an individual carrying a germ line MEN1 mutation (6,7). Menin, a predominantly nuclear protein (8,9), is widely expressed from early in fetal development onwards. Several strains of Men1 knock-out mice have been generated, both constitutive and conditional in specific tissues. Heterozygous Men1 ϩ/Ϫ mice from about 9 months of age develop hyperplasia and tumors of parathyroid and anterior pituitary glands and pancreatic islets (10 -12). This mouse model closely mimics human MEN1. Tissue-specific knockouts of the Men1 gene have been generated in which null expression of Men1 in parathyroids, the pituitary, and pancreatic islet ␤-cells has been achieved (13)(14)(15)(16). These studies suggest that although loss of menin contributes to the neoplasias that eventually arise, it does not affect normal development in these particular tissues.
Mice that are constitutively homozygous null for the Men1 gene die in utero at mid-gestation of multiple defects in developing organs, suggesting that menin does play critical essential roles in tissues additional to those affected in the MEN1 syndrome (10,17,18). The fetuses are small and exhibit craniofacial defects, suggesting that menin might play a role in both endochondral and intramembranous bone formation (10,17). We previously showed that menin is required for the commitment of multipotential mesenchymal stem cells in vitro to the osteoblast lineage (19). This occurs in part by menin facilitating BMP-2 signaling via Smads and the transcriptional activity of the key osteoblast transcription factor, Runx2 (20). Menin interacts physically and functionally with Smads1/5 and Runx2 in mesenchymal stem cells (20). In committed osteoblasts these interactions are for the most part lost, as menin then acts to maintain the osteoblast in its differentiated state. This is mediated in part by menin's interaction with the TGF-␤/Smad3 pathway (20,21).
Based on our previous in vitro studies, we hypothesized that menin plays a critical role not only in osteoblastogenesis and osteoblast differentiation but also in bone mass accumulation in vivo (22). To further understand the role of menin in bone development in vivo we generated and examined mouse models in which the expression of the Men1 gene is specifically altered only in mature osteoblasts.

Generation of Knock-out and Transgenic Mice
Conditional knock-out and transgenic mouse models in which either the Men1 gene is deleted or the menin cDNA is overexpressed specifically in differentiated osteoblasts were generated. Mice were maintained in a pathogen-free standard animal facility, and experimental procedures were performed following an animal use protocol approved by the Animal Care and Use Committee of McGill University.

Col1a1-Menin-Tg Transgenic mice
A full-length FLAG-tagged human menin cDNA preceded by a rabbit ␤-globin intron was inserted in between a 2.3-kb mouse Col1a1 promoter fragment (24) and a SV40 polyadenylation signal. The transgene sequence was released from the plasmid backbone by SacII restriction enzyme digestion. Transgenic founders were generated by pronuclear injections at McGill Cancer Center Transgenic Core Facility. For osteoblast-specific menin transgenic mice analysis, their wild-type littermate mice were used as controls. The mutant mouse strain of Col1a1-Menin-Tg mice was on the C57BL6/J background.

Osteoblast-specific Men1 Knock-out and Meninoverexpressing Transgenic Mice
Genotyping strategies are available upon request.

Col1a1-Menin-Tg Mice
The OC-Cre transgene was detected using the primers 5Ј-CAAATAGCCCTGGCAGATTC-3Ј and 5Ј-TGATACA-AGGGACATCTTCC-3Ј to generate a 260-bp product corresponding to a portion of the OC promoter and the rabbit ␤-globin intron.
Reverse transcription (RT)-polymerase chain reaction (PCR) was performed to detect the expression of the Col1a1-Menin transgene in tissues. See "Gene Expression Analysis" below for RNA extraction and cDNA synthesis. A forward primer specific for human menin (5Ј-GTGAGAAGATGAAGGGCATGA-3Ј) and a reverse primer specific for the SV40 polyadenylation signal (5Ј-TAATGTGGCTATGGGAATTGGA-3Ј) amplified a 640-bp transgene product. As an internal standard, a 350bp product of the hypoxanthine phosphoribosyltransferase (HPRT) cDNA was amplified using specific primers; F (5Ј-GTTGAGAGATCATCTCCACC-3Ј) and R (5Ј-AGCGAT-GATGAACCAGGTTA-3Ј). The PCR conditions were 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min for 30 cycles.

Immunoblotting
Tissues and isolated primary osteoblasts were homogenized on ice in triple detergent buffer, and Western blotting was performed as previously described (25) with anti-menin polyclonal antibody, Ab2605 (Abcam), or anti-FLAG M2 antibody (Sigma). Membranes were reprobed with a ␤-tubulin antibody (Santa Cruz) as the loading control.

Bone Mineral Density (BMD) Analysis by Dual Energy X-ray Absorptiometry
Anesthetized mice were placed prone on a specimen tray in a PIXImus densitometer (Lunar Corp., Madison, WI), densitometry was performed on the right femur, and BMD was calculated with the manufacturer's software (version 1.46.007).

Micro-computed Tomography (Micro-CT)
Mice femurs, dissected free of soft tissue, were fixed overnight in 70% ethanol. High resolution images of the distal femur were acquired with a desktop microtomographic imaging system (SkyScan 1072, Antwerp, Belgium) in accordance with the recommendations of the American Society of Bone and Mineral Research (26). Three-dimensional renderings of the distal 3.5 mm of the femora were reconstructed using two-dimensional data from scanned slices with the 3D creator software (Skyscan). The trabecular bone region of interest was drawn to include all cancellous bone in the metaphysis.

Bone Histomorphometric Analysis
Lumbar vertebrae were dissected, fixed for 24 h in 4% formalin, dehydrated in a graded ethanol series, and embedded in methyl methacrylate resin, and 7-m sections were made (27). Von Kossa/van Gieson staining was done, and histomorphometric analyses were made with Osteomeasure software (OsteoMetrics Inc.). The parameters comply with the guidelines of the American Society of Bone and Mineral Research nomenclature committee (28). For analysis of osteoblasts, osteocytes, and osteoclasts, 5-m sections were stained with toluidine blue and tartrate-resistant acid phosphatase, respectively. Images were taken at room temperature using a light microscope (DM200; Leica) with a 20ϫ (numerical aperture of 0.40) or 40ϫ (numerical aperture of 0.65) objective. All histological images were captured using a camera (DP72; Olympus), acquired with DP2-BSW software (XV3.0; Olympus), and processed using Photoshop (Adobe).

TUNEL Assay
To evaluate osteoblast apoptosis the TUNEL assay was performed on undecalcified vertebral sections (see above) using the Deadend Fluorometric TUNEL System kit (Promega). Images were captured on an EVOS FL Digital Inverted Fluorescent Microscope (Fisher).

Double Calcein Labeling
Calcein (Sigma; C-0875) was dissolved in buffer (0.15 M NaCl, 2% NaHCO 3 ) and injected twice intraperitoneally (25 g/g body weight) at 9 and 2 days before the mice were killed (29). Bones were harvested and embedded in plastic as described above. Serial sections were cut, and the freshly cut surface of each section was viewed and imaged using fluorescence microscopy. The double calcein-labeled width was measured using Osteomeasure software, and the mineral apposition rate (MAR ϭ interlabel width/labeling period), bone formation rate/bone surface (BS), and mineralizing surface (MS)/BS were calculated. Calculations were made on a minimum of duplicate specimens from replicate mice of each group.

Osteoclastogenesis Assay
Bone marrow cells were collected from mouse long bones under aseptic conditions as described previously (30) and were cultured overnight with M-CSF (25 ng/ml Preprotech; 300 -25). The following day non-adherent precursors were plated at 5 ϫ 10 4 cells/cm 2 and treated with M-CSF (50 ng/ml) and recombinant soluble RANKL (50 ng/ml) (31) for 5 days after which osteoclast numbers were quantified (see below).

Fluorescence Labeling
Co-cultures on glass coverslips were fixed on day 9 with 10% formalin, permeabilized with 0.1% Triton X-100 (Fisher; BP151-100) in PBS for 10 min, incubated for 20 min at room temperature with a red fluorescent Alexa Fluor 568 tagged phalloidin (Invitrogen; A12380), washed 3 times in PBS and once with distilled water, and incubated with 0.2 g/ml of 40,6diamidino-2-phenylindole dihydrochloride (DAPI) in distilled water for 5 min. Slides were washed three times with distilled water and mounted on microscope slides using mounting media (Thermo Scientific; 9990402). Images were recorded using an inverted fluorescence microscope (Eclipse TE2000-U, Nikon) and cooled charge-coupled device camera (Hamamatsu Photonics) supported by Volocity software (Perkin Elmer) (32).

Osteoblast Isolation from Young Mice
Osteoblasts from calvaria of individual 1-2-week-old OC-Cre;Men1 f/f pups or their control littermates were isolated as described (33). The bones were dissected free of sutures and subjected to 2 consecutive digestions at 37°C with shaking in ␣MEM medium (Invitrogen) containing 0.1 mg/ml collagenase P (Roche Applied Science) and 0.25% trypsin/0.1% EDTA (Invitrogen), The supernatant was discarded leaving the pieces of bone. Fresh digestion medium containing 0.2 mg collagenase P/ml was added, and calvaria were incubated at 37°C with vigorous shaking every 15 min for 45 min or until bone pieces began to fall apart. The bone pieces and cells were collected by centrifugation (1500 ϫ g), washed with ␣MEM, and plated in a 10-cm dish with ␣MEM containing 10% FBS and 1% penicillin/ streptomycin (Invitrogen). After 4 days, the medium was changed, and when the cells reached 80% confluency they were passaged to 6-well plates (10 5 cells/well). After reaching ϳ70% confluency, the medium was supplemented with 10 mM ␤-glycerophosphate and 50 g/l ascorbic acid (Sigma), and this osteogenic medium was replaced every 3 days.

Osteoblast Isolation from Older Mice
Osteoblasts from calvaria of individual 5-6 month-old OC-Cre;Men1 f/f mice or their control littermates were isolated as described (34). Calvaria were dissected and transferred to a Petri dish containing PBS, and soft tissues were removed by scraping the bone with a scalpel. Sutures were removed, and the rest of the calvaria was chopped into small fragments. The fragments were incubated for 30 min at 37°C with shaking in 2 mg of collagenase II/ml ␣MEM (Bioshop Inc), the supernatant was discarded, and the procedure was repeated. Calvaria were then incubated for 30 min at 37°C with shaking in a 0.25% trypsin, 0.1% EDTA solution. The supernatant was discarded and replaced by fresh collagenase II solution for the fourth and final incubation step of 30 min at 37°C. Bone pieces were washed 3 times with ␣MEM containing 10% FBS and 1% penicillin/streptomycin, 10 mM ␤-glycerophosphate, and 50 g/l ascorbic acid ("osteogenic medium") and transferred to T75 flasks. Medium was changed every 3 days. Adult mouse bone cells migrated from the bone chips after 2-3 days, and after 7 days the cells were passaged to 6-well plates (10 5 cells/well).

Adenovirus Infection of Osteoblasts
Osteoblasts were isolated from calvaria of neonatal and adult Men1 f/f mice as above. They were grown to 70% confluency and infected with control adenovirus expressing green-fluorescent protein (ad-GFP) or adenovirus expressing Cre recombinase (ad-Cre, Vector Biolabs) at an multiplicity of infection of 100 (35). Forty-eight hours after adenoviral infection, cells were replated for proliferation and differentiation assays.

Gene Expression Analysis in Osteoblasts
For gene expression analyses, RT-PCR was performed. Total RNA was prepared from primary osteoblasts in 6-well plates using the standard TRIzol method as recommended by the manufacturer (Invitrogen) and subjected to DNase I (Invitrogen) treatment. Semiquantitative RT-PCR was performed to estimate the abundance of specific mRNAs relative to GAPDH mRNA. Two g of total RNA was employed for the synthesis of single-stranded cDNA using a high capacity cDNA reverse transcription kit (Applied Biosystems). Primer sequences and cycle numbers are available on request.

ALP Activity and Mineral Apposition
Confluent primary cultures of osteoblasts were grown in differentiation osteogenic medium. ALP activity was determined by histochemical staining at days 7-10 post-differentiation using the Fast Red TR/Naphthol AS-MX tablet set (Sigma). Cells were washed with PBS then fixed at room temperature for 10 min using 100% ethanol. After removal of the ethanol, cells were washed with distilled water, and stain was added. Fixed cells were incubated at 37°C for 30 min then washed with running water and air-dried.
Phosphate deposition was evaluated in osteoblasts 10 days post-differentiation. Osteoblasts were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Fixed cells were washed 5 times with distilled water Ͼ30 min then stained with von Kossa solution (3% silver nitrate in distilled water) under bright light. Cells were washed before scanning or photographing.
Calcium deposition was determined by alizarin red-S staining. Cells were rinsed with PBS, fixed for 10 min in 100% ethanol at room temperature, washed with distilled water, then stained for 30 min with 4 mM alizarin red-S, pH 4.2. Alizarin red-S was extracted by destaining with 10 mM HCl in 70% ethanol, and mineral accumulation was quantified on a microplate reader at 520 nm. The cell layer was then solubilized in lysis buffer (10 mM formamide, 50 mM sodium acetate, 1% SDS, pH 6), and protein content was assessed by a Bradford assay. Results were calculated as nmol of alizarin red-S/mg of protein and expressed as -fold change versus control.
The Men1 gene deletion is specific for bone.
Methyl Thiazolyl Tetrazolium (MTT) Dye Viability Assay Primary osteoblasts were seeded at a density of 4 ϫ 10 4 cells per well in 24-well tissue culture plates and cultured in phenol red-free DMEM-F-12 at 37°C. Forty-eight hours later MTT was added, and incubation was continued for 4 h. Acid isopropyl alcohol was added, and absorbance was read at 595 nm.
PrestoBlue Cell Viability Assay-Primary osteoblasts were seeded at a density of 4 ϫ 10 4 cells per well in 24-well tissue culture plates and cultured in DMEM-F-12 at 37°C. Forty-eight hours later, PrestoBlue reagent was added, and incubation was continued for 20 min. Absorbance was read at 570 nm and normalized to the 600-nm value.

Osteoblast Apoptosis
Mouse C2C12 cells and adult primary osteoblasts (Men1 f/f or OC-Cre;Men1 f/f ) were seeded at a density of 1 ϫ 10 6 in 60-mm plates. After 48 h cells were treated or not with 1 mM dexamethasone and incubated for a further 24 h. Proteins in cell lysates were separated by SDS-PAGE, transferred to PVDF membranes and Western-blotted overnight at 4°C with anticaspase-3 antibody (ABCAM, ab47131) followed by HRP-conjugated secondary antibody. Detection was performed using enhanced chemiluminescence.

Serum Biochemistries
Mouse blood samples were obtained by cardiac puncture, and serum biochemistries were determined by autoanalyzer at the Comparative Medicine Animal Resources Centre of McGill University. .02 .03 .04 .05 .06 .07 .08 .09  Table 1 for quantitation. Deletion of osteoblast Men1 leads to reduction in trabecular bone assessed by histomorphometry. D, representative images of von Kossa/van Gieson staining of vertebrae of 9-month-old OC-Cre;Men1 f/f and Men1 f/f control mice. See Table 1 for quantitation.

Statistical Analysis
Results are expressed as the mean Ϯ S.E. Statistical comparisons using GraphPad Prism Version 4.00 analysis software (GraphPad Software, Inc., San Diego, CA) were made using the unpaired two-tailed Student's t test, and a p value of Ͻ0.05 was taken to indicate a significant difference.

Disruption of the Men1 Gene Is Specific to the Bone-PCR
amplification of tissue genomic DNA confirmed that Cre-mediated recombination occurred specifically in bone of the knock-out, OC-Cre;Men1 f/f , mice (Fig. 1). Body weights (g) (

Osteoblast-specific Disruption of the Men1 Gene Decreases
Bone Mass and Volume-BMD determined by dual energy x-ray absorptiometry of the femurs of 9-month-old mice was significantly reduced in knock-out mice relative to controls ( Fig. 2A). OC-Cre;Men1 f/f mice had significant decreases in both trabecular and cortical bone by micro-CT. Trabecular bone volume was markedly reduced in the knock-out mice (Fig. 2B), and the trabeculae were of abnormal structure (Fig.  2C). Bone volume and surface, trabecular number, and cortical bone thickness were significantly reduced in OC-Cre; Men1 f/f mice (Table 1). Structure model index, trabecular thickness, and trabecular separation were significantly increased (Table 1).
Histomorphometric analysis indicated that trabecular bone volume and surface at lumbar spine was remarkably reduced in OC-Cre;Men1 f/f mice compared with controls ( Fig. 2D; Table   1). These combined data show that trabecular and cortical bone mass were reduced, and trabecular structure was altered in OC-Cre;Men1 f/f mice.

Deletion of the Men1 Gene in Osteoblasts Decreases Bone
Formation and Turnover-Cancellous MAR, bone formation rate/ BS, and MS/BS were significantly decreased in OC-Cre;Men1 f/f mice compared with controls (Fig. 3A, i-iv). In addition, the number of osteoblasts was significantly reduced in OC-Cre; Men1 f/f mice relative to controls (Fig. 3B, i and ii). The apoptotic rate of osteoblasts in the menin knock-out OC-Cre;Men1 f/f mice was increased versus control mice assessed by TUNEL assay (Fig. 3B, iii and iv). Moreover, the osteoclast number was also significantly reduced in OC-Cre;Men1 f/f mice compared with control mice (Fig. 3C, i and ii). These findings suggest that osteoblast menin plays an important role in bone formation as well as bone turnover and is critical for maintenance of normal bone remodeling. In addition, osteocyte numbers were increased in OC-Cre;Men1 f/f mice relative to controls (Fig. 3D,  i and ii).
Osteoblast-specific Deletion of the Men1 Gene Decreases Osteoclastogenesis-Multinucleated osteoclasts formed from the bone marrow of OC-Cre;Men1 f/f mice and cultured with exogenous M-CSF and RANKL were no different in appearance and number from those of Men1 f/f mice (Fig. 4A, i and ii). However, in co-culture experiments in which osteoclastogenesis relies on the endogenous factors produced by osteoblasts formed by treatment with ascorbic acid a clear difference was observed. First, osteoblast formation assessed by alkaline phosphatase staining was reduced in the marrow co-cultures of the OC-Cre;Men1 f/f mice relative to those of Men1 f/f mice (Fig. 4B,  i and ii). Second, osteoclastogenesis was significantly reduced, as assessed by total TRAP-positive cells, in the menin osteoblast knock-out mice relative to that in control mice (Fig. 4C, i and  ii). Finally, multinucleated osteoclasts quantified in fluorescently labeled cells were markedly deficient in the marrow cocultures of the osteoblast menin knock-out mice versus those of the control mice (Fig. 4D, i and ii).
Hence, although the generation of osteoclasts from the bone marrow cells of the knock-out OC-Cre;Men1 f/f mice was unimpaired when exogenous M-CSF and RANKL were added, the bone marrow cells of the knock-out mice demonstrated markedly deficient osteoclastogenesis when only ascorbate was added to stimulate osteoblast differentiation without added osteoclastogenic factors.
Primary Cultures of Calvarial Osteoblasts of Neonatal and Adult Mice-Primary osteoblasts were isolated from individual calvaria of 2-week-old (neonatal) and 6-month-old (adult)   (Fig. 5A, i and ii) and BrdU assay (Fig. 5B, i and ii), primary calvarial osteoblasts of the adult OC-Cre;Men1 f/f mice proliferated more rapidly than those of the Men1 f/f controls. By Alizarin Red-S staining for calcium deposition (Fig. 5C, i and ii), von Kossa staining for phosphate deposition (Fig. 5Ci) primary calvarial osteoblasts of the adult OC-Cre;Men1 f/f mice were markedly impaired with respect to mineral apposition relative to those of the Men1 f/f control mice. Likewise, the primary calvarial osteoblasts of the adult OC-Cre;Men1 f/f mice were markedly impaired with respect to ALP staining (Fig. 5Ci) and activity (Fig. 5Ciii) compared with those of the Men1 f/f control mice.
Menin-deficient Osteoblasts Undergo Caspase-3 Cleavage More Readily Than Wild Type-To assess the ability of the menin-deficient osteoblasts to undergo apoptosis, we examined the extent of procaspase-3 cleavage promoted by dexamethasone. Control mouse C2C12 cells or Men1 f/f or OC-Cre; Men1 f/f calvarial osteoblasts were stimulated or not with 1 M dexamethasone for 24 h. Cell extracts were subjected to SDS-PAGE and Western blot analysis with anti-caspase-3 antibody. Whereas the dexamethasone treatment clearly stimulated cleavage of pro-caspase-3 and production of the active caspase-3 17-kDa fragment in the control C2C12 and the OC-Cre;Men1 f/f , cells this was not apparent in the control osteoblasts (Fig. 5D). Hence, the menin-deficient osteoblasts are more susceptible to apoptotic stimuli than the control osteoblasts. This result is consistent with the TUNEL assay results on vertebral sections showing enhanced apoptotic rate of osteoblasts in the menin knock-out versus control mice (Fig. 3B, iii and iv).

Altered Expression of Genes Involved in Osteoblast Differentiation and Proliferation, Osteoclast Formation, Osteocyte Formation, and Apoptosis-To examine the effect of osteoblast
Men1 loss on gene expression, semi-quantitative RT-PCR was performed on RNA from isolated osteoblasts from the Men1 knock-out and control mice. The expression of key osteoblast marker genes for BMP-2, Runx2, Osterix, Dlx5, Col1a1 and osteocalcin, were reduced in osteoblasts from adult OC-Cre; Men1 f/f mice relative to those from control mice (Fig. 6B) rectly, of the BMP/TGF-␤ signaling pathways. Although the expression of both OPG and RANKL was decreased in osteoblasts from adult OC-Cre;Men1 f/f mice relative to controls, the relative decrease for RANKL was greater, favoring an increase in the OPG/RANKL ratio (Fig. 6C). This would be consistent with the finding of reduced numbers of osteoclasts in the OC-Cre;Men1 f/f mice (Fig. 3C, i and ii) as well as the impaired support of osteoclastogenesis observed in the co-culture assays with bone marrow of the osteoblast menin knock-out versus control mice (Fig. 4, A-D). Expression of TGF-␤ target genes important for cell cycle and/or differentiation control was also altered, being either down-regulated (p15, p21, p18, p27) or up-regulated (CDK2 and CDK4) in OC-Cre;Men1 f/f osteoblasts (Fig. 6D). These results are consistent with the finding that osteoblasts from OC-Cre;Men1 f/f mice proliferated more rapidly relative to controls (Figs. 5B, i and ii). Expression of osteocyte markers, Sclerostin, Phex, and DMP-1, was higher in osteoblasts from OC-Cre;Men1 f/f mice relative to controls (Fig.  6E). Expression of proapoptotic markers Bax and Bad was Primary calvarial Men1 f/f osteoblasts were infected with adenovirus expressing GFP (control) or Cre recombinase. A, mineralization was assessed by Alizarin Red-S assay. i, staining at 21 days after culture in osteogenic medium. ii, quantitation. ***, p Ͻ 0.001; Adeno-Cre versus Adeno-GFP. B, RNA was isolated, semiquantitative RT-PCR and gel electrophoresis was performed, and densitometric analysis was made. Expression of osteoblast markers Runx2, Osx, and osteocalcin was reduced, the cyclin-dependent kinase inhibitor p27 was reduced, CDK4 was increased, osteocyte markers sclerostin and DMP-1 were increased, pro-apoptotic marker Bad was increased, and anti-apoptotic marker Bcl-2 was decreased. **, p Ͻ 0.01; ***, p Ͻ 0.001; Adeno-Cre versus Adeno-GFP. ϪRT, minus reverse transcriptase control. C, tissue protein (calvaria, femur, cartilage, and kidney) was extracted, and a Western blot was conducted with antibodies against FLAG (Col1a1-Menin-Tg transgene), menin (endogenous and exogenous menin), and ␤-tubulin (loading control). The transgene (menin) was expressed specifically in bone of the Col1a1-Menin-Tg mice.

Hprt
higher, whereas that of the anti-apoptotic Bcl-2 was lower (Fig.  6E). Taken together with the in vivo findings, the osteoblast gene expression data would suggest that the menin-deficient osteoblasts are functionally defective but proliferate more rapidly to the point at which they become osteocytes or undergo apoptosis. The reprogramming of the osteoblast gene expression profile occurs early as identical changes to those in the adult osteoblasts were found in neonatal osteoblasts of the OC-Cre;Men1 f/f mice relative to controls (data not shown).

Disruption of Men1 in Primary Calvarial
Osteoblasts in Vitro-To assess the effect of directly disrupting the osteoblast Men1 gene in vitro, primary adult Men1 f/f osteoblasts were infected with either Adeno-Cre or Adeno-GFP. The Adeno-Cre-infected cells in which menin expression was reduced by Ͼ70% relative to the Adeno-GFP-infected controls demonstrated markedly reduced mineralization by Alizarin Red-S assay (Fig. 7A, i and ii). Gene expression analysis by semiquantitative RT-PCR indicated decreases in osteoblast markers, increases in osteocyte markers, and altered proliferation and apoptotic genes (Fig. 7B). These changes were all similar to the findings in primary calvarial osteoblasts of the OC-Cre;Men1 f/f mice relative to controls (Figs. 5C and 6, B-E).
Specific Osteoblast Overexpression of Menin Increases Bone Mass Accumulation-To overexpress Men1 specifically in osteoblasts in vivo, we made a Col1a1-Menin transgene construct in which a 2.3-kb Col1a1 promoter fragment drives a human FLAG-tagged menin cDNA (Fig. 8A). Pronuclear injection of the construct into fertilized mouse eggs resulted in seven founders of which two demonstrated bone-specific expression of menin. The mice were then mated with wild-type mice. Col1a1-Menin-Tg mice generated from this breeding were analyzed for transgene expression using semiquantitative reverse transcriptase PCR, showing that the transgene was expressed only in bone tissue (Fig. 8B). The overexpression of menin in bone was confirmed by Western blot analysis (Fig. 8C).
Col1a1-Menin-Tg mice were no different from controls with respect to weight (g) ( Table 2.  (Fig. 9, A and B). There were significant increases in bone volume and surface and trabecular numbers as well as decreases in structure model index and trabecular spacing (Fig. 9C, i-iv; Table 2). Furthermore, von Kossa/van Gieson staining of lumbar vertebrae indicated that trabecular bone was increased in Col1a1-Menin-Tg mice compared with controls (Fig. 9D). By histomorphometric analysis, bone volume and surface were significantly increased in Col1a1-Menin-Tg mice ( Fig. 9D; Table 2). Cancellous MAR was significantly increased in Col1a1-Menin-Tg mice compared with control mice (Fig. 10A, i and ii), and the number of osteoblasts was significantly increased in Col1a1-Menin-Tg mice (Fig. 10B, i and ii), although the numbers of osteoclasts (Fig.  10C, i and ii) and osteocytes were not altered (Fig. 10D, i and ii). Therefore, to a large extent the bone phenotype of the Col1a1-Menin-Tg mirrors that of the OC-Cre;Men1 f/f mice.

Effect of Menin Overexpression in the Osteoblast on Proliferation, Differentiation, Mineralization, and Gene Expression-
Primary calvarial osteoblasts were isolated from adult Col1a1-Menin-Tg mice. The Col1a1-Menin-Tg osteoblasts demonstrated reduced numbers in cell viability MTT and Prestoblue assays (Fig. 11A, i and ii), increased mineralization by Alizarin Red-S assay (Fig. 11B, i and ii), and increased alkaline phosphatase differentiation marker activity (Fig. 11C, i and ii). Therefore, the phenotype of the Col1a1-Menin-Tg primary calvarial osteoblasts largely mirrors that of the OC-Cre;Men1 f/f mice osteoblasts. By semiquantitative RT-PCR, osteoblast differentiation markers, Runx2, osteocalcin, and Col1a1, were increased, as were CDKIs, p15, and p21, whereas CDK4 was decreased (Fig. 11D). These changes mirrored those in the OC-Cre; Men1 f/f mice osteoblasts. Expression of RANKL, OPG, and Sclerostin as well as Bax and Bcl-2 were unchanged in the Col1a1-Menin-Tg primary osteoblasts relative to controls (Fig.  11D). These data are consistent with the unchanged osteoclast and osteocyte numbers and suggest that susceptibility to apoptosis is unaltered. The fact that overexpressing menin did not affect osteoclast-modulating, osteocytic, and apoptosis-related genes whereas deleting menin increased these genes suggests that there is a threshold at which the level of menin is sufficient for an effect on a particular gene. Therefore, the data suggest that the thresholds are different for different genes (and importantly for different subsets of genes).

DISCUSSION
We investigated the role of menin in bone by using a Cre-mediated recombination strategy to conditionally disrupt the  FEBRUARY 13, 2015 • VOLUME 290 • NUMBER 7 gene in osteoblasts as well as overexpressing menin specifically in osteoblasts. We show that menin is important for proper osteoblast function and maintenance of bone mass in vivo.

Osteoblast Menin and Bone
The tumor suppressor menin is encoded by the MEN1 gene in which germ line mutations cause the autosomal dominant MEN1 disorder characterized by the development of tumors in select endocrine (and non-endocrine) tissues (6,7). Menin is widely expressed from early in fetal development. Heterozygous constitutional knock-out Men1 ϩ/Ϫ mice closely mimic their human MEN1 counterparts and from 9 months of age develop hyperplasia and tumors of parathyroid and anterior pituitary glands and pancreatic islets (10 -12). Mice having tissue-specific knock-out of the Men1 gene in parathyroid, pituitary, or pancreatic ␤-cells develop normally, suggesting that menin does not influence development of these tissues (13)(14)(15)(16). However, constitutively null Men1 Ϫ/Ϫ mice die in utero at midgestation of multiple defects in developing organs, suggesting that menin is critical for normal development (10,17,18). The fetuses are small and have craniofacial defects suggesting that menin might play a role in both endochondral and intramembranous bone formation (10,17). We showed previously that menin promotes the commitment of mesenchymal stem cells in vitro to the osteoblast lineage (19). This occurs in part by the roles played by menin in facilitating BMP-2 signaling via Smads and transcriptional activity of the key osteoblast regulator, Runx2 (20). Menin interacts physically and functionally with Smads1/5 and Runx2 in mesenchymal stem cells. In committed osteoblasts, menin appears to restrain the osteoblasts in their differentiated state, and this is achieved in part by menin's interaction with the TGF-␤/Smad3 pathway (20).
In mesenchymal stem cells in vitro inactivation of menin only affects the BMP-2-induction of osteoblasts and apparently has little effect on generation of chondrocytes or adipocytes (19). In addition, others have shown that by modulating responsiveness to the cytokines TGF-␤ and BMP-2, menin functions to shift the balance of multipotential mesenchymal cell commitment to the osteogenic rather than the myogenic lineage (36). In vivo deletion of the Men1 gene in Pax3-or Wnt1-expressing neural crest cells (that give rise to many cell lineages including the mesenchymal stem cells that become osteoblasts) leads to perinatal death, cleft palate, and other cranial bone defects (37). Values are the mean Ϯ S.E. (n ϭ 3 independent experiments). *, p Ͻ 0.05. C, ALP. Primary osteoblasts were cultured in an osteogenic medium containing ␤-glycerophosphate and L-ascorbic acid for 7 and 14 days (shown). i, ALP staining. Washed, fixed cells were overlaid with nitro blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3Ј-indolyl phosphate p-toluidine salt) (NBT) and incubated at room temperature for 20 min in the dark. ii, ALP activity. p-Nitrophenyl phosphate substrate was added to lysed cell extracts, and the reaction was stopped with 3 N NaOH. Absorbance was read at 405 nm, and values were normalized to protein concentration. Values are the mean Ϯ S.E. (n ϭ 3 independent experiments). *, p Ͻ 0.05; **, p Ͻ 0.01. D, RNA was isolated, semi-quantitative RT-PCR and gel electrophoresis were performed, and densitometric analysis was made. Expression of osteoblast markers Runx2, osteocalcin, and Col1a1 was increased, OPG and RANKL were unaltered, the CDKIs p15 and p21 were increased, CDK4 was decreased, and the osteocyte marker Sclerostin, the pro-apoptotic Bax, and the pro-survival Bcl-2 were unaltered. *, p Ͻ 0.05; **, p Ͻ 0.01 Col1a1-Menin-Tg versus control.
Deletion of menin in Pax3-expressing somite precursors also produces patterning defects of rib formation (37).
In the present studies we have taken the direct approach of deleting the Men1 gene, on the one hand, and of overexpressing menin, on the other, in the osteoblast. This has provided validation that menin is important in vivo for osteoblast function and maintenance of bone mass. The Men1 osteoblast knockout mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with their control littermates. Osteoblast and osteoclast numbers as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but exhibited deficient mineral apposition and reduced alkaline phosphatase activity. In contrast to the knock-out mice, transgenic mice overexpressing osteoblast menin showed a gain of bone mass, and osteoblast number and mineral apposition rate were increased relative to their control littermates.
The gene expression analysis of osteoblasts in vitro provided insights into alterations that underlie the in vivo phenotype of the Men1 knock-out mice. Promoter-reporter assays demonstrated that the menin-deficient osteoblasts were impaired with respect to their ability to respond normally at the transcriptional level to either TGF-␤ or BMP-2. This was consistent with the finding of reduced expression of key osteoblast markers such as BMP-2 itself and Runx2, Osterix, Dlx2, Dlx5, and osteocalcin. These genes are all under the control, either directly or indirectly, of the BMP-2/TGF-␤ signaling pathways. As a consequence, in vivo osteoblast number, bone formation, and bone mass were reduced.
Production of the bone resorbing cell, the osteoclast, is stimulated by the RANKL synthesized by the osteoblast that acts via cell-cell contact on RANK expressed by the osteoclast precursor (38). The relative expression of the decoy receptor, OPG, secreted by the osteoblast, is also key in dictating the final outcome with respect to osteoclast generation (39). Although the expression of both OPG and RANKL, was decreased in menindeficient osteoblasts, that of RANKL was greater, favoring an increase in the OPG/RANKL ratio. This would be consistent with the finding of reduced numbers of osteoclasts in the osteoblast Men1 knock-out mice and their decreased ability to support osteoclastogenesis in the bone marrow co-culture assays. As bone formation and bone resorption are coupled (40), the findings identify impaired bone remodeling as an additional underlying cause of the reduced bone volume in the Men1 osteoblast knock-out mice.
Expression of genes important for cell cycle control was also altered, with that for CDKIs, p15, p18, p21, p27, being down regulated, whereas that for CDK2 and CDK4 being up-regulated. This was all consistent with the proliferation rate of the menin-deficient osteoblasts being greater than those of the controls. Osteocyte markers (Phex, DMP-1, and Sclerostin) were increased, and pro-apoptosis markers (Bax and Bad) were also increased, whereas that of the anti-apoptotic marker, Bcl-2, was decreased. These findings are consistent with the finding of increased numbers of osteocytes in the Cre-OC; Men1 f/f mice, on the one hand, and the enhanced apoptotic rate of osteoblasts observed in vivo as well as the enhanced suscep-tibility of primary calvarial Cre-OC;Men1 f/f osteoblasts in vitro to undergo apoptosis as indicated by a caspase-3 cleavage assay on the other hand. Given the fact that menin is required for the ability of BMPs to exert their effects on osteoblast differentiation via a Smad-dependent pathway (19,20), it might be unexpected that menin-deficient osteoblasts exhibit more (rather than less) apoptosis. However, the present data are fully consistent with previous findings that BMP-2 promotes osteoblast apoptosis through a Smad-independent pathway (41).
Taken together with the in vivo findings, the gene expression data support the notion that the functionally defective osteoblasts proliferate to reach the point at which they either undergo apoptosis or become osteocytes much more quickly than normal. Hence, in vivo, osteoblast and osteocyte numbers are reduced and increased, respectively. Therefore, a lack of menin leads to a marked reprogramming of the osteoblast gene expression profile with subsequent phenotypic changes.
In summary, Men1 osteoblast knock-out mice displayed a significant reduction of bone mass with decreases in osteoblast number, bone formation rate, and bone remodeling. On the other hand, bone volume was increased and bone formation was stimulated in transgenic mice overexpressing menin in the osteoblast. These findings suggest that menin is required for optimal bone formation and maintenance of bone mass. Primary osteoblasts from the Men1 knock-out and the meninoverexpressing mice had altered gene expression profiles with respect to signaling pathways under the control of BMPs and TGF-␤. In conclusion, menin, or more specifically the menin/ Smad interface, can be considered as a potential gain of function therapeutic target for treatment of low bone mass disorders. The desired compounds would be those that mimic the effect of menin (as a transcriptional facilitator) upon the BMP receptor-regulated Smads.